Antimicrobial therapy has focussed on antibiotics that affect processes unique to bacteria, such as compositions that affect enzymes and components of the cell wall (e.g., penicillin), and prokaryotic ribosome inhibitors (e.g., streptomycin). To a lesser extent, antibiotic therapy has also exploited structural and catalytic differences between enzymes common to prokaryotes and eukaryotes.
Antimicrobial or antibiotic therapy is dependent upon the discovery of biochemical systems that are unique to bacteria and which can be safely inhibited--i.e., systems which can be inhibited without producing detrimental effects or undesired side effects in or upon the individual receiving such therapy. Further, it has been observed that antibiotic resistance increases in pathogen populations due to recruitment of resistance enzymes from the microbial gene pool, partially as a result of antibiotic overuse or misuse. As resistance develops, it has become increasingly difficult to identify unique biochemical pathways which may be inhibited in bacteria, which are not also represented in the cells of higher organisms, including man.
Therefore, one goal of the present disclosure is the revelation of a new, apparently ubiquitous biochemical and regulatory system unique to bacteria, which may be exploited for antimicrobial therapy. Unique regulatory proteins are described herein which are absent from eukaryotic cells and could provide novel targets for antimicrobial therapy.
One exemplary "target" regulatory system involves bacterial protein kinases. Histidine protein kinase plays an important role in bacterial signal transduction. Typically, histidine protein kinase activity is assayed via a two-step procedure including a phosphorylation reaction in the presence of [.gamma.-.sup.32 P]ATP followed by SDS-PAGE and autoradiography analysis. Application of this method in large-scale screening for histidine protein kinase inhibitor is limited, however, largely because of the need to use SDS-PAGE.
The regulation of biological activities of proteins by reversible phosphorylation plays an important role in control of cellular response to extracellular stimuli in both prokaryotic and eukaryotic organisms. Phosphorylation cascades mediated by bacterial two-component systems provide a conserved mechanism for coordinate regulation in response to signal input. In bacteria, diverse processes such as chemotaxis (Hess, et al., PNAS USA 84: 7609-7613 (1987); Wylie, et al., Biochem. Biophys. Res. Commun. 151: 891-896 (1988); Hess, et al., Cell 53: 79-87 (1988)), nitrogen starvation (Ninfa and Magasanik, PNAS USA 83: 5909-5913 (1986); Keener and Kotsu, PNAS USA 85 4976-4980 (1988); Weiss and Magasanik, PNAS USA 85: 8919-8923 (1988)), osmotic regulation (Aiba, et al., J. Biol. Chem. 264: 8563-8567 (1989); Forst, et al., PNAS USA 86: 6052-6056 (1989); Igo, et al., Genes & Dev. 3: 589-605 (1989)), sporulation (Perego, et al., J. Bacteriol. 171: 6187-6196 (1989)), and certain types of antibiotic resistance (Christopher, Science 261: 308-309 (1993); Guenzi, et al., Mol. Microbiol. 12: 505-515 (1994)) are regulated by a two-component system.
In general, two-component systems comprise a sensor protein (usually a histidine protein kinase) and a response regulator protein. The histidine protein kinase undergoes ATP-dependent autophosphorylation on a histidine residue in response to a stimulus. The phosphorylated sensor protein then transfers the phosphor group to an aspartyl residue of the response regulator protein, which protein either acts as a transcriptional regulator or interacts with another protein.
Inhibition of either the autophosphorylation or the subsequent phosphor-transfer by special inhibitors of the two-component system would interrupt the signal transduction pathway, thereby providing a means to interfere with a particular cellular process. Inhibitors specific to the bacterial two-component system are of particular importance in the development of new antibacterial or antibiotic agents.
An efficient assay system is necessary for large-scale screening of inhibitors of the two-component system. The conventional in vitro assay of the two-component system involves a phosphorylation reaction of histidine protein kinase and its substrate in the presence of [.gamma.-.sup.32 P]ATP. The histidine protein kinase, the response regulator protein, and unincorporated [.gamma.-.sup.32 P]ATP are then separated by SDS-PAGE followed by autoradiographic analysis (Burbulys, et al., Cell 64: 544-552 (1991)). Although this conventional assay provides a sensitive measurement of histidine protein kinase activity, the throughput of the assay is very limited due to the SDS-PAGE step. Other separation techniques, such as trichloroacetic acid precipitation and HPLC are not suitable for the two-component system because of the instability of aspartyl phosphate (Burbulys, et al., Id. (1991)). Therefore, the assay systems and methods disclosed herein are particularly useful and overcome the deficiencies of other available methods.
An efficient assay system for histidine protein kinase has now been developed in which the substrate is immobilized onto Ni-resin via a six-histidine tag (or linker). In this assay system, the separation of the substrate from the kinase and [.gamma.-.sup.32 P]ATP is achieved by removal of the reaction mixture from the resin, and the extent of phosphorylation of the substrate is then determined by measuring the radioactivity remaining in the resin.
The data presented herein demonstrate a good and highly reproducible correlation between kinase activity, as measured by the extent of phosphorylation of the substrate and the radioactivity remaining on the resin. This assay system has been adapted into a high throughput screening assay using an automated liquid handling system and 96-well filter plates. This has made it possible to process in excess of six 96-well plates per operator per day.
The within-disclosed high throughput assay for histidine protein kinase is particularly useful for inhibitor screening purposes. An assay system for histidine protein kinase activity without SDS-PAGE separation or acid precipitation is also described. Bacillus subtilis KinA (histidine protein kinase) and SpoOF (cognate response regulator) are used as kinase and substrate, respectively, in the within-described assays. It should be appreciated, however, that the disclosed assay systems can also be applied to other protein kinases and their substrates. Kinetic features of the within-disclosed assays and the adaptation of an assay system into a high throughput assay for histidine protein kinases are also presented herein.